comparative study of methanol, butyrate, and hydrogen as electron donors for long-term...

Comparative Study of Methanol, Butyrate,and Hydrogen as Electron Donors forLong-Term Dechlorination ofTetrachloroethene in MixedAnerobic Cultures

Federico Aulenta,1 James M. Gossett,2 Marco Petrangeli Papini,1

Simona Rossetti,3 Mauro Majone1

1Department of Chemistry, University of Rome ‘‘La Sapienza,’’ P.le Aldo Moro 5,00185 Rome, Italy; telephone: þ39-06-49913716; fax: þ39-06-490631;e-mail: [email protected] of Civil and Environmental Engineering, Cornell University,Ithaca, New York 148533Water Research Institute, National Research Council (IRSA-CNR),Via Reno 1, 00198 Rome, Italy

Received 15 December 2004; accepted 15 March 2005

Published online 8 July 2005 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bit.20569

Abstract: This study examined the ability of differentelectron donors (i.e., hydrogen, methanol, butyrate, andyeast extract) to sustain long-term (500 days) reductivedechlorination of tetrachloroethene (PCE) in anerobic fill-and-draw bioreactors operated at 3:1 donor:PCE ratio(defined on a total-oxidation basis for the donor). Initially(i.e., until approximately day 80), the H2-fed bioreactorshowed the best ability to completely dechlorinatethe dosed PCE (0.5 mmol/L) to ethene whereas, in thepresence of methanol, butyric acid or no electron donoradded (but low-level yeast extract), dechlorination waslimited by the fermentation of the organic substrates andin turn by H2 availability. As the study progressed, theH2-fed reactor experienced a diminishing ability todechlorinate, while more stable dechlorinating activitywas maintained in the reactors that were fed organicdonors. The initial diminished ability of the H2-fed reactorto dechlorinate (after about 100 days), could be partiallyexplained in terms of increased competition for H2

between dechlorinators and methanogens, whereasother factors such as growth-factor limitation and/oraccumulation of toxic and/or inhibitory metabolites wereshown to play a role for longer incubation periods (over500 days). In spite of decreasing activity with time, the H2-fed reactor proved to be the most effective in PCEdechlorination: after about 500 days, more than 65% ofthe added PCE was dechlorinated to ethene in the H2-fed

reactor, versus 36%, 22%, and <1% in the methanol-fed,butyrate-fed, and control reactors, respectively.� 2005 Wiley Periodicals, Inc.

Keywords: competition; electron donors; ethene; long-term dechlorination; PCE

INTRODUCTION

Enhanced in situ anaerobic reductive dechlorination (RD) is

a promising technology for remediation of tetrachloroethene

(PCE)-contaminated groundwater (Morse et al., 1997). In

situ enhanced RD can be accomplished by stimulating the

activity of native dechlorinating populations through the

addition of electron donors to provide the electrons required

for PCE or trichloroethene (TCE) reduction. Recent studies

have indicated that the selection of electron donor(s) may

impact the ability to sustain the RD activity in situ

(Ballapragada et al., 1997; Carr and Hughes, 1998; Fennell

et al., 1997; Smatlak et al., 1996). Several different electron

donors, including methanol, butyrate, lactate, and benzoate

(Carr andHughes, 1998;DiStefano et al., 1991; Fennell et al.,

1997; Yang and McCarty, 1998) have been shown to support

enhanced RD of PCE, both in field and in laboratory studies.

Nevertheless, in most of the cases, the hydrogen produced

during fermentation of organic compounds was the actual

electron donor used for the RD. Indeed, although there is

recent evidence that some halorespiring bacteria can use

acetate as electron donor (He et al., 2002), H2 is typically the

direct electron donor for this process (DiStefano et al., 1992;

Maymo-Gatell et al., 1995).

�2005 Wiley Periodicals, Inc.

Correspondence to: Federico Aulenta

Contract grant sponsors: Ministero dell’Ambiente e della Tutela del

Territorio; National Research Council, CNR (Gruppo Nazionale per la

Difesa dai Rischi Chimico Industriale Ecologici, GNDRCIE)

Contract grant number: PR.3.29/URM

One major concern regarding the choice of fermentable

organic substrates is the possible competition for hydrogen

that can establish between dechlorinators and other H2-

utilizingmicroorganismssuchasmethanogens (Fennell et al.,

1997; Yang and McCarty, 2002). However, previous studies

demonstrated that dechlorinators have the potential to out-

compete other H2-utilizers when H2 is present at low

concentration, due to dechlorinator’s higher affinity for

hydrogen (i.e., its lower half-saturation constant and lower

threshold for hydrogen use) (Ballapragada et al., 1997;

Smatlak et al., 1996; Yang and McCarty, 1998). Conse-

quently, the use of substrates which are slowly fermented,

and only under low hydrogen partial pressures, would result

in a competitive advantage to dechlorinators over other

H2-using microorganisms. Recently, complex organic com-

pounds have been used in laboratory experiments as

slowly fermenting, low-cost donors for the RD of PCE

(DiStefano et al., 2001; Kao et al., 2003; Yang and McCarty,

2002; Yu and Semprini, 2002).

A possible drawback with use of fermentable organic

substrates to stimulate in situ the RD of chlorinated solvents

is the production and accumulation in the subsurface of large

amounts of fermentation products, such as acetate or

propionate, and resulting deterioration of groundwater

quality.

In principle, direct hydrogen addition may offer some

advantages over the use of slowly fermentable organics. For

instance, when a low number of H2-producing fermentative

microorganisms is present at a site, the dechlorination rate

can be accelerated by directly providing dechlorinating

microorganisms with hydrogen. In addition, hydrogen does

not leave any environmentally harmful residue in the

subsurface, is generally less expensive than most organic

compounds, and generates less biomass in the subsurface so

its use is less likely to modify groundwater flow patterns

(ESTCP, 2002).

Thus, technologies based on direct hydrogen addition to

the subsurface have been recently developed, based on

passive dissolution through membranes (Clapp et al., 2004;

Fang et al., 2002; Ma et al., 2003), low-pressure sparging

(Newell et al., 1997), or hydrogen-generating electrodes

(Zhang et al., 2001). However, several aspects may still limit

the use of direct hydrogen addition to support the RD of

chlorinated solvents—its poor solubility in water and its

tendency, once injected in the subsurface, to rapidly escape

from the contamination plume. Moreover the presence of

high levels of H2 could also provide a selective advantage to

methanogens and eventually result in the marginalization of

dechlorinators. As an example, Ma et al. (2003) utilized a

polyethylene hollow-fiber membrane to deliver hydrogen in

soil columns. Even though the membrane-supplied H2

effectively stimulated PCE dechlorination, the system was

very inefficient in that only 5% of the supplied H2 was used

for dechlorination.Most of the remainderwas used to support

methanogenesis (94%). In addition, extensive growth of

methanogens eventually resulted in excessive foulant

accumulation on the outside of the membrane. Aside from

issues of hydrogen-competition and direct biomass-induced

fouling, bubble formation from excessive, subsurface

methanogenesis can significantly diminish the hydraulic

conductivity of an aquifer, and can result in explosive levels

of methane that pose safety concerns (Fennell and Gossett,

2003). Furthermore, most isolated dechlorinators, including

Dehalococcoides spp., while using hydrogen as electron

donor for the reduction of the chlorinated solvents, require

acetate as carbon source for growth (De Wildeman et al.,

2003; He et al., 2002; Maymo-Gatell et al., 1997) and the

presence of specific (and in some cases not yet identified)

growth factors. For instance, DiStefano et al. (1992) observed

that H2-utilizing dechlorinators have nutritional dependency

on the metabolic products of other organisms in a methanol-

fed anerobic culture. Similar results were reported for pure

cultures of Dehalococcoides ethenogenes; even though this

microorganism uses H2 as its sole electron donor, it needs

unknown growth factors that must be provided by other

fermentative bacteria (Maymo-Gatell et al., 1997).

Despite the extensive number of publications reporting the

use of different electron donors to stimulate theRDof PCEby

microbial cultures, there are conflicting results on which

donors are more efficient. Additionally, very few studies

examined the effect on process stability of long-term enrich-

ment on different donors. The latter issue is relevant since

bioremediation technologies are generally designed for long-

term operation.

The aim of this study was to compare methanol, butyrate,

and hydrogen for their ability to sustain the long-term (i.e.,

500-days) reductive dechlorination of PCE.

MATERIALS AND METHODS

Bioreactor Operation

In this study four completely mixed, suspended-growth,

PCE-dechlorinating bioreactors were operated in a fill-and-

draw mode for 500 days. The bioreactors consisted of glass

bottles (total volume 0.56 L, liquid volume 0.35 L), sealed by

rubber stoppers, screw caps, and mixed continuously with

magnetic stirring bars. Each reactor was initially seeded with

the supernatant (50 mL) of a different brackish sediment

microcosm from Venice Lagoon (Italy). The different

microcosms had been preliminarily enriched, in the presence

of the sediment (300 g dry weight bottle-1) on PCE and each

substrate for a period of approximately 6months. During this

period, every 14 days, the microcosms received a dose of

PCE (0.25 mmol/L, as nominal concentration—i.e., total

amount added to the bottle divided by the liquid volume), a

dose of the selected electron donor—i.e., none, methanol

(1 mmol/L), butyrate (0.3 mmol/L), or hydrogen (3 mmol/L,

as nominal concentration), and a dose of yeast extract

(resulting in a microcosm concentration of 10 mg/L). Before

each re-feeding, the reactors were purgedwith 70%N2–30%

CO2 to remove volatile compounds (including any residual

chlorinated ethenes as well as ETH and methane). All the

microcosms developed the ability to dechlorinate PCE

744 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 91, NO. 6, SEPTEMBER 20, 2005

completely to ETH. At the end of this 6-month enrichment

period, 50 mL of supernatant was removed from each

microcosm and used as a seed to inoculate the (sediment-

free) suspended-growth reactors. By adopting this seeding

methodology, the initial biomass concentrations (as volatile

suspended solids, VSS) in the reactors were: 90 mg/L for

the Me-reactor; 69 mg/L for the Bu-reactor; 83 mg/L for the

H2-reactor; 47 mg/L for the Control.

The suspended-growth reactors were maintained at

25.0� 0.58C in a water bath. Every 7 days, each reactor

received a dose of PCE (0.5 mmol/L, as nominal concentra-

tion—i.e., total amount added to the bottle divided by the

liquid volume), a dose of the selected electron donor—i.e.,

none, methanol (2 mmol/L), butyrate (0.6 mmol/L), or

hydrogen (6 mmol/L, as nominal concentration), and a dose

of yeast extract (resulting in a reactor concentration of 10mg/

L). PCE, methanol, and butyric acid were added in neat form

by using glass syringes; hydrogen was added in the

headspace of the reactor by using gas-tight syringes. Yeast

extract was added from an anoxic, aqueous stock solution by

glass syringe.

The electron donors (i.e., methanol, butyric acid, hydro-

gen) were added to provide the same amount of reducing

equivalents (i.e., 12 meq/L, based on their complete

oxidation)—threefold in excess to that required for the

complete reduction of the added PCE to ETH. Yeast extract

(10 mg/L) was also added to all the reactors as a nutritional

supplement to simulate the possible influence of a complex

mixture of organic substrates, often present in contaminated

aquifers, on the reductive dechlorination of PCE.

The reactor that was fed methanol as the primary electron

donor for PCE dechlorination is hereafter referred to as

Me-reactor; the reactor that was fed butyric acid is referred to

as Bu-reactor; the reactor that was fed hydrogen is referred to

as H2-reactor; the reactor to which no electron donor was

added, but yeast extract at 10 mg/L, is referred to as Control.

Before each re-feeding, the reactorswere purgedwith 70%

N2–30% CO2 to remove volatile compounds (including any

residual chlorinated ethenes as well as ETH and methane),

and 17.5 mL of suspended culture was withdrawn and

replaced by fresh, reduced, basal medium. The resulting

constant PCE and electron donor volumetric loads were

571 and 1,714meq/L/day respectively.With this procedure an

average hydraulic and biomass retention time of 140 days

was maintained.

At day 251, before the usual weekly feeding, the H2-

reactor was supplemented with a filtered supernatant from

theMe-reactor as a possible source of growth factors. For this

experiment, the Me- and H2-reactors were transferred inside

the anerobic glovebox, then 35 mL of supernatant was

removed with a sterile 50-mL syringe from the Me-reactor,

immediately filtered (0.45 mm-pore size-filter) to remove

microorganisms, and then added to the H2-reactor.

At day 338, the liquid phase in all the reactorswas replaced

by fresh medium, by using an ultrafiltration cell (Amicon

8400). This operation consisted in pouring the contents of

each reactor into the ultrafiltration cell (in an H2-free

anaerobic glovebox); concentrating the cell suspension

(350 mL) into approximately 25 mL, and then resuspending

in fresh basal medium to a final volume of 350 mL. This

operation allowed removal of solutes with a molecular

weight less than the membrane’s molecular-weight

cutoff (i.e., 100,000 Da) while retaining the cells within the

system.

Basal Medium Composition

The basal medium contained (final concentration in grams

per liter): (NH4)2HPO4, 0.62; K2HPO4, 0.4; MgHPO4, 0.06;

CaCl2 �H2O, 0.04; resazurin 0.001; and 10 mL of a trace

metal solution (Zeikus, 1977). After preparation, themedium

was dispensed into a 250 mL serum bottle, which was then

sealed with a Teflon1-coated stopper (Wheaton, Millville,

NJ). Subsequently, the bottle was flushed with a 70% N2–

30% CO2 gas mixture to remove dissolved oxygen and

received the following additions (per 100 mL of medium):

1 mL vitamin solution (Balch et al., 1979), 0.5 mL

Na2S � 9H2O 5% w/v, and 2 mL NaHCO3 10% w/v.

Analytical Procedures

Volatile components [PCE, TCE, DCE (dichloroethene), VC

(vinyl chloride), ETH, CH4] were quantified by injecting

50 mL of reactor headspace (with a gas-tight syringe) into a

gas chromatograph equipped with flame-ionization detector,

as described previously (Aulenta et al., 2002). Methanol was

quantified by injecting 1 mL of an aqueous sample into a

Perkin Elmer 8400 gas chromatograph (2 m� 2 mm glass

column packed with 60/80 mesh Carbopak1 B/1% SP-1000

Supelco; N2 carrier gas 30mL/min; oven temperature 1108C;flame-ionization-detector temperature 2608C). Butyrate,

propionate and acetate were determined by injecting 1 mLof aqueous sample into the Perkin Elmer 8400 (2 m� 2 mm

glass column packed with 80/120 mesh Carbograph1 1AL,

Alltech; N2 carrier gas 30 ml/min; oven temperature 1758C;flame-ionization-detector temperature 2008C). H2 was

determined by injecting 0.5 mL of reactor headspace gas

into a Varian 3400 GC (stainless-steel column packed with

molecular sieve, Supelco, N2 carrier gas 18 mL/min; oven

temperature 1808C; thermal-conductivity detector (TCD)

temperature 2008C).Standards for PCE, TCE, cis-dichloroethene (cis-DCE),

VC, ETH, CH4, and H2 were prepared by adding a known

amount of each compound to a serum bottle with the same

headspace-to-liquid ratio as the reactor (Gossett, 1987). It

was assumed that DCE produced from more highly

chlorinated ethenes was cis-DCE, which has a different

calibration constant from trans-DCE due to having a higher

Henry’s constant (Gossett, 1987). This assumption is

supported by previous work with the same cultures, in the

presence of the sediment, which indicated that 1,1-DCE and

trans-DCEwere not produced in appreciable amounts during

PCE dechlorination (data not reported). Detection limits for

PCE, TCE, and cis-DCE were ffi10 mM; for VC, ETH, and

AULENTA ET AL.: EFFECT OF DIFFERENT E-DONORS ON LONG-TERM DECHLORINATION OF PCE 745

CH4 were ffi0.1 mM; for H2 was ffi10 mM; for acetate,

methanol, butyrate, and propionate were ffi2 mg/L.

Chemicals

Neat PCE (99þ%), TCE (99.5þ%), and cis-DCE (97%) were

purchased from Aldrich Chemical, Co. (Milwaukee, WI). VC,

ETH, hydrogen, and methane gases (99.9þ%) were purchased

from Scott Specialty Gases (Bellefonte, PA). Methanol

(99.9þ%), Butyric acid (99%), and yeast extract were

purchased by Aldrich Chemical. All the other chemicals used

to prepare analytical standards or feed solutionswere purchased

from Aldrich Chemical, Co. and were analytical grade.

Batch Dechlorination Assays

Two different types of batch dechlorination assays were

carried out in this study using the reactors themselves. The

first type (type-1) consisted in following the time-course of

PCE dechlorination, and in some cases of the electron-donor

utilization, during a normal, 7-day, feeding cycle of the

reactor. This experiment allowed comparison of the different

electron donors in their ability to sustain the dechlorination of

PCE under usual feeding conditions. Type-1 batch assays

were repeated several times during the fill-and-draw period,

so to follow the evolution of the different cultures. Type-1

batch assays were also carried out immediately before and

immediately after the ultrafiltration (UF) and resuspension of

cells in fresh medium.

In the second type of batch assay (termed type-2), H2

(12 meq/L) was spiked to the reactors in place of the normal

electron donor. Except for the change in electron donor

added, experiments were performed identically to those

described above. Since preliminary results (Aulenta et al.,

2002) indicated thatH2was the actual electron donor for PCE

dechlorination, these experiments allowed determination of

the maximum dechlorinating activity under conditions of

unlimited electron-donor and electron-acceptor concentra-

tions (Aulenta et al., 2004). As for type-1 assays, type-2

assays were also replicated at different times to follow the

evolution of the cultures.

Calculations

The reducing equivalents channeled to dechlorination,

methanogenesis, and acetogenesis during type-1 and type-2

batch experiments were calculated from the measured

concentrations of dechlorination products, methane, and

acetate formed (DiStefano et al., 1991; Gao et al., 1997).

Molar equivalents factors used were: 8 eq/mol for methane,

8 eq/mol for acetate, and 2 eq/mol for each chlorine removed

from chlorinated ethenes. The reducing equivalents available

from the degradation of yeast extract were estimated assum-

ing it has the typical chemical composition of biomass (i.e.,

C5H7O2N). From this assumption it can be calculated that

1.8meq/Lwould be available from the complete oxidation of

10 mg/L yeast extract.

RESULTS

Kinetics of PCE Dechlorination With the DifferentElectron Donors and With Excess Hydrogen

Figure 1 shows, for the different reactors, the time-course of

PCE dechlorination under usual electron-donor conditions

(type-1 batch assay, days 70–76) or with excess H2 (type-2

batch assay, days 77–83).

Under usual feeding conditions (days 70–76), all of the

electron donors enhanced PCE dechlorinationwith respect to

the Control (Fig. 1). In the presence of methanol (Fig. 1B),

PCE was rapidly consumed in less than 2 days and converted

to DCE, VC, and ETH with little intermediate accumulation

of TCE. Thereafter little and slow transformation of the

chlorinated intermediates occurred. This was undoubtedly

due to the depletion ofmethanol (data not shown). Also in the

Bu-reactor (Fig. 1C), PCE was completely exhausted in less

than 2 days and mainly converted to DCE (with only little

accumulation of TCE). However, at that time butyric acid

was still present and sustained further dechlorination of DCE

to VC and ETH. In the H2-reactor (Fig. 1D) PCE was very

rapidly dechlorinated to VC (in less than 1 day). Thereafter,

VC dechlorination to ETH proceeded, although at slower

rate, and the complete dechlorination of the dosed PCE to

ETH was achieved in about 3 days.

At day 77 (the feeding cycle immediately following that

above described), a dechlorination batch experiment with

excess H2 was carried out on the dechlorinating reactors

(type-2 batch assay, Fig. 1A). In Me-, Bu-, and Control-

reactors, PCE dechlorination commenced without any initial

lag and proceeded far faster than in the presence of the usual

substrates (i.e., methanol, butyric acid, or no electron donor

added, respectively). In the H2-reactor, which did not

experience any change of electron donor, the time-course

of PCE dechlorination was almost identical to that observed

in the previous cycle (days 70–77).

The addition of H2 to the control reactor resulted in the

transformation of the dosed PCE to VC (through inter-

mediate accumulation of DCE) and, to a much lesser extent,

ETH. Also in theMe- and Bu-reactors, the direct H2 addition

increased both the rate and the extent of PCE dechlorination.

For both reactors, the complete transformation of the dosed

PCE to ETH was observed in about 4 days.

The results of these experiments provided strong indica-

tion that: (a) H2 was the actual donor of reducing equivalents

for the RD of PCE for the microbial consortia enriched on

yeast extract, methanol, or butyric acid; and (b) in the

presence of these substrates the dechlorination of PCE was

limited by the availability of hydrogen produced during the

fermentation of these organic electron donors.

Figure 2 shows the cumulative dechlorination curves (i.e.,

reducing equivalents routed to dechlorination vs. time) for

the batch dechlorination assays described in Figure 1. For

type-1 batch assay, the initial cumulative dechlorination rate

with H2 (299 meq/L/h) (as calculated from the regression of

the initial linear time profile of the cumulative dechlorination


curve) was much higher than with methanol (73 meq/L/h), orwith butyric acid (66 meq/L/h), or without electron donor

added (11 meq/L/h). Conversely, in the presence of excess

hydrogen (type-2 batch assay), the initial cumulative

dechlorinating activity in the H2-reactor (320 meq/L/h) wasonly slightly higher (20%–30%) than that observed in the

Me-reactor (i.e., 260 meq/L/h) and in the Bu-reactor (i.e.,

256 meq/L/h). On the other hand, it was approximately six

times higher than that observed in the Control-reactor (i.e.,

35 meq/L/h). This seems to indicate that after 76 days of

operation the H2-, Me-, and Bu-reactors showed a similar

maximum dechlorinating potential and possibly a compar-

able concentration of dechlorinating biomass. On the other

hand, a significantly lower concentration of dechlorinating

biomass was presumably present in the Control.

Long-Term Dynamics of theDechlorinating Bioreactors

The evolution of PCE dechlorination in the four bioreactors

was followed by monitoring the performance of the reactors

over long time.Major changeswere noticed in theH2-reactor,

which experienced a progressively diminishing ability to

dechlorinate. Figure 3 shows the time-course of VC

(produced from PCE dechlorination) in successive feeding

cycles, starting from day 85 to 110. As shown in Figure 3, the

VC produced from PCE dechlorination was no longer being

completely dechlorinated to ETH within the 7-day feeding

cycle [whereas higher chlorinated ethenes (i.e., PCE, TCE,

cis-DCE) were still completely removed]. The last step of

PCE dechlorination (i.e., from VC to ETH) was more

adversely affected than VC formation. This period of rapid

decrease of RD activity also corresponded to the onset of an

intense methanogenic activity (inset in Fig. 3).

Coupled type-1 and type-2 batch assays were replicated at

different times along thewhole fill-and-drawperiod (days 98,

244, 320, 340, and 462 for type-1 batch assays; days 105, 230,

and 469 for type-2 batch assays)—Figure 4A and B. It is

evident that other reactors did not exhibit the quick decrease

Figure 1. Time course of PCEdechlorination during two successive feedings.Day 70–77: PCEþ normal electron donor (type-1 batch dechlorination assay);

day 77–84: PCEþ excess hydrogen (type-2 batch dechlorination assay).A: Control; (B)Me-reactor; (C) Bu-reactor; (D) H2-reactor. Symbols: (~), PCE; (&),

TCE; (&), cis-DCE; (*) VC; (�), ETH.

Figure 2. Time course of dechlorination (in terms of cumulative electron

equivalents) for the batch experiments described in Figure 1. Symbols: (~),

H2-reactor; (&), Me-reactor; (*), Bu-reactor; (�), Control.


of RD rate shown by the H2-reactor within 98–105 days. On

the other hand, by 230–244 days all reactors showed a

marked decrease of RD rate both in type-1 and type-2 batch

assays.

To investigate if the more rapid decrease of RD rate in the

H2-reactor was due to the lack of nutrients or growth factors,

at day 251 it was supplemented with 35 mL of filtered

(0.45 mm) supernatant from the Me-reactor (see ‘‘Bioreactor

Operation’’ above). The latter was chosen because of itsmore

stable RD activity over time. Subsequently, a type-1 batch

assay was carried out on the H2-reactor: maximum

dechlorination rate increased of less than 10% with respect

to the previous feeding cycle (Fig. 4A).

Considering that a diminished RD activity was observed,

over time, for all the reactors, at day 340, the liquid phasewas

almost fully renewed with fresh basal medium by using an

ultrafiltration cell (UF) (see ‘‘Experimental’’). Following

liquid-phase renewal, a type-1 batch assay was performed

and showed a clear increase of RD rate for all reactors

(Fig. 4A). This suggests that metabolic intermediates and/or

end products might have accumulated in the mixed liquor to

inhibiting levels, or else that some factor in the basal medium

was limiting (and that addition of fresh medium aided

dechlorination).

Thereafter, the usual feeding procedurewas continued and

at days 430–469 a new set of assays was carried out. Again,

after a long period (i.e., 90–130 days after the ultrafiltration)

all reactors showed a decrease of RD rate, and this decrease

was most evident for the H2-reactor. After 469 days of

operation, the Me-, and Bu-reactors showed higher max-

imum dechlorination rates than the H2-reactor in type-2

assays (Fig. 4B). In other words, after 469 days of operation,

the maximum dechlorinating potential of the Me-, and Bu-

reactors was largely superior to that of the H2-reactor which

was comparable to the Control.

However, even after 469 days of operation, the maximum

dechlorination rates of these reactors in the presence of their

respective carbon sources remained lower than rates

observed when H2 was added to each. This means that the

potential RD performance remained limited by H2 avail-

ability from fermentation for the entire experimental period.

Usage of Electron Donors

Throughout the experimental period, methanol was never

detected at the end of the feeding cycles in the Me-reactor.

Type-1 batch assays showed that methanol was quickly

consumed at an average rate of 40.8� 20.2 mmol/L h

(average value �90% confidence intervals). Usually more

than two-thirds of the reducing equivalents available from

methanol consumption were channeled to acetate, with the

remaining being used in the reductive dechlorination

(Fig. 5B). Methane formation was a minor sink for reducing

equivalents available from methanol (less than 5% of

methanol consumption on reducing equivalent basis) for

most of the experimental period.

Butyric acid degradation appeared to be very slow

(2.6� 2.0 mmol/L h, average value �90% confidence

intervals); occasionally it was not completely consumed

within the 7-day period between additions. In the Bu-reactor,

acetate formationwas themajor sink for reducing equivalents

Figure 3. Profile of VC in successive feedings with PCE (0.5 mM) and H2

(6 mM) in the H2-reactor. Insert: Methane and VC concentrations at the end

of the feeding cycles in the period between day 50 and 110.

Figure 4. Observed maximum dechlorination rate for the dechlorinating

reactors when fed the normal electron donors (type-1 batch dechlorination

assay) (A), and excess hydrogen (type-2 batch dechlorination assay) (B).Type-1 batch assays were carried out at days 70, 98, 244, 320, 340, and 462.

Type-2 batch assays were carried out at days 77, 105, 230, 469.


available from butyric acid, whereas reducing equivalents

channeled to dechlorination were generally between 15%

and 25% (Fig. 5C). Propionate was occasionally detected as

intermediate of butyric acid degradation, but never accumu-

lated at high concentrations (data not shown). Throughout the

study, in the Bu-reactor methane formation was always

negligible.

In the H2-reactor, the dose of H2 was routinely consumed

within the initial 2–3 days of the cycle (data not shown), with

an average consumption rate of 124.7� 10.8 mmol/L h

(average value �90% confidence intervals). As shown in

Figure 5D, up to 30% of hydrogen was diverted to reductive

dechlorination. Until day 60, negligible methane accumu-

lated at the end of the feeding cycles, thereafter methane

formation rapidly increased (inset in Fig. 3). By day 100, till

the end of the experimental period, a stable methane

formation was present in the reactor which accounted by

for up to 80% of available hydrogen (Fig. 5D). Indeed,

approximately 8 meq/L of methane were routinely measured

at the end of each feeding cycle. It could not be ascertained

whether this methane production was due to acetotrophic or

hydrogenotrophic activity. Homoacetogenic activity was

also observed. In the Control no primary electron donor was

added except yeast extract at 10mg/L. Themain constituents

of yeast extract could not be identified and therefore could not

be measured in this study. Apparently, very efficient

utilization of reducing equivalents available from yeast

extract was observed in the Control (Fig. 5A). Acetate was

found to be the only other product of yeast extract

degradation (Fig. 5A). In Figure 5A, recoveries over 100

suggest that reducing equivalents available fromyeast extract

were probably underestimated (see experimental). Through-

out the study, negligible methane formation was observed in

this reactor.

Amount of PCE Dechlorinated

Figure 6A–D shows, for all the reactors, the PCE added on a

cumulative basis, and the PCE dechlorination products

formed. The mass-balance of (chloro)ethenes in terms of

cumulative sum of PCE dechlorination products formed

is also displayed (dashed line). Figure 6 clearly indicates

that, despite the observed diminished ability to dechlorinate,

over the 500-day period of operation, the H2-reactor

dechlorinated more PCE, based on the cumulative dechlor-

ination products formed, than other reactors, with more

than 65% of the added PCE that was dechlorinated to

ETH. In fact, 209 meq/L were routed to dechlorination in the

H2-reactor at the end of experimental period, while 160 meq/

L in the Me-reactor, 134 meq/L in the Bu-reactor, and

84 meq/L in the Control. Figure 6 also shows that good mass

balances of (chloro)ethenes (i.e., PCE recovered into

dechlorination products, onmolar basis) could bemaintained

during the whole experimental period, ranging from

Figure 5. Distribution (%) of added reducing equivalents toward the different metabolisms.A: Control; Since it was not possible tomonitor the consumption

of yeast extract, for the purpose of this figure it was assumed that added yeast extract was completely consumed within each 7-day feeding cycle. Reducing

equivalents available from the complete oxidation of yeast extract were estimated assuming yeast extract with the typical chemical composition of biomass

C5H7O2N; (B) Me-reactor; (C) Bu-reactor; (D) H2-reactor. For Me-, Bu-, and H2-reactors the reducing equivalents provided by yeast extract were accounted

assuming that the added yeast extract was consumed within each 7-day feeding cycle.


85.9%� 6.5% for the Control to 109.6%� 8.9 (%) for the

Me-reactor.

DISCUSSION

Initially (i.e., until approximately day 80), the maximum

dechlorinating activity (type-2 assay, under unlimited H2 and

PCE concentrations) was similar between the H2-, Me-, and

Bu-reactors indicating that the reactors contained compar-

able amount of dechlorinating biomass. Nevertheless, under

usual feeding conditions (type-1 assay) the rate of PCERD in

the Me- and Bu-reactor proceeded at much slower rate than

the H2-reactor, being probably rate-limited by the fermenta-

tion of the organic substrates and in turn by H2 availability.

By around 90 days, the H2-reactor had experienced a

noticeable decline in dechlorination ability—most notice-

ably in the VC-to-ETH step. The Me- and Bu-reactors also

experienced a progressive decrease in dechlorination poten-

tial (type-2 assay), but this occurred later (at ca. 230 days) and

to a lesser degree. On the other hand, by the end of the

experimental period, the reducing equivalents routed to

dechlorination in the H2-reactor were 1.3 times those routed

to dechlorination in theMe-reactor, 1.6 times those in the Bu-

reactor, and 2.5 times those in the Control reactor.

In large part, these results may reflect that the amount of

free hydrogen produced from fermentation of butyrate or

methanol was much lower than theoretical ratio of 3:1 eq/eq

based on total oxidation. In the H2-reactor, all equivalents

(12 meq/L) resulting from the direct addition of H2 were

potentially available to the H2-utilizing dechlorinators;

whereas in the case of the organic substrates, only the H2

produced from their fermentation could be rapidly or initially

utilized by dechlorinators. Butyrate fermentation to acetate,

for example, yields only 2H2 (or 4 eq of H2) per mole of

butyrate; therefore, expressed on the basis of expected

hydrogen produced from primary fermentation of butyrate,

our dose rate of 0.6 mmol/L would have produced only

2.4 meq/L of H2—far less than the 12 meq/L of hydrogen

added directly to the H2-reactor and insufficient, even, to

handle the 4 meq/L of PCE added. A similar estimate of H2

from methanol is not possible since methanol conversion

does not necessarily result in a net H2 production; conversion

of methanol to H2/CO2 is only energetically favorable at low

hydrogen concentrations and therefore depends on the

relative activity of methanol-fermenters and H2-users (e.g.,

dechlorinators, methanogens, sulfate reducers) (Balk

et al., 2002; Cord-Ruwish and Ollivier, 1986; Goorissen

et al., 2004). In the absence of fast and efficient H2-

consumption methanol-fermenters will mostly produce

acetate instead of H2/CO2.

To be sure, in the cases of both butyrate-fed and methanol-

fed reactors, biomass (grown from the directly fed donors as

well as from their fermentation products such as acetate)

Figure 6. Long-term performance of the anaerobic PCE-dechlorinating reactors: cumulative PCE additions (—); cumulative TCE (&); DCE (&); VC (*);

and ETH (�) production. The cumulative molar sum of chloroethenes and ethene present at the end of each feeding cycle (� � �) is also displayed. A: Control;(B) Me-reactor; (C) Bu-reactor; (D) H2-reactor.


would contribute significant, endogenous, secondary sources

of hydrogen in subsequent, complex fermentative decay of

biomass. However, these subsequent, secondary reducing

equivalents from a donor such as butyrate—i.e., beyond the

initial 2.4 meq of H2/liter—might be expected to be only

slowly and incompletely available to dechlorination.

Hence, the finding that in the Bu-reactor dechlorination

usually accounted for 15%–25% of electrons available from

total oxidation of butyrate indicates that dechlorinating

microorganisms could effectively compete for the H2

available from butyrate fermentation. In addition, the amount

of reducing equivalents available from butyrate fermentation

that were channeled toward RD was usually in very good

agreement with the theoretical amount of H2 available from

butyrate fermentation. Similarly, in the Me-reactor, no more

than 25% of electrons potentially available from methanol

degradation could be scavenged for RD of PCE, with the

remaining being diverted to acetate.

Negligible methanogenic activity developed in the Me- or

Bu-reactors, whereas a significant methanogenic activity

developed in the H2-reactor, which accounted for most (up to

80%) of the electrons available from the added H2. This was

observed despite the high PCE concentrations adoptedwhich

were found to completely inhibit methanogenic activity in

previous studies (DiStefano et al., 1991). Previous research

also indicated that PCE-dechlorinating cultures fed H2 at

high concentrations would eventually fail as methanogens

came to predominate the culture and dechlorinators were

marginalized (Fennell et al., 1997). Thiswas likely the reason

for the diminished ability of the H2-reactor to dechlorinate

that was observed after the initial 90 days (see Figs. 3 and 4).

On the other hand, from day 100 to 500, the maximum

methane formation rate remained approximately constant at

200 meq/L/h (data not shown), whereas maximum dechlor-

ination rate by the H2-reactor significantly decreased (from

300 to less than 70 meq/L/h).Alternatively, the higher sensitivity of the H2-reactor

to long-term enrichment may have been due to the

occurrence of nutrient deficiencies or to the accumulation

of toxic and/or inhibitory metabolites. The first hypothesis is

supported by previous studies. For instance, DiStefano et al.

(1992) found that in order to sustain the dechlorination of

PCEwithH2 for periods longer than 40 days, it was necessary

to supplement the cultures with filtered supernatant from a

methanol-fed culture. The authors concluded that H2-

utilizing dechlorinators have nutritional dependency on the

metabolic products of other organisms in the methanol-fed

system. Later studies (Maymo-Gatell et al., 1995) elucidated

that the apparent superiority of methanol compared to

hydrogen in sustaining the dechlorination of PCE was due

to the fact that methylotropic methanogens and acetogens

growing on methanol are rich in cobamides such as vitamin

B12, which could be possibly cross-fed, through lysis or

excretion, to the dechlorinating microorganisms; Dehalo-

coccoides spp. were found to require large amounts of

vitamin B12 (up to 0.05 mg/L) to dechlorinate. In our study,

vitamin B12 was provided at a significantly lower concentra-

tion (i.e., 0.002 mg/L). On the other hand, the amount

supplied was that typically supplied to organisms which use

vitamin B12 for anabolic reactions (Cote and Gherna, 1994).

Providing the dechlorinating microorganisms with poten-

tially suboptimal amounts of vitamin B12 allowed investiga-

tion of whether their nutritional requirements could be

otherwise met by microorganisms growing on the different

substrates tested.

On the other hand, only little beneficial effect on the

dechlorinating activity of the H2-reactor was observed when it

was supplementedwith 35mLoffiltered (0.45 mm) supernatant

from the Me-reactor (Fig. 4A). This suggests that the filtered

supernatant did not apparently contain any beneficial growth

factor that was lacking in the H2-reactor; however, given the

B12-poor basal medium employed here, it would be likely that

most of the B12 in the systemwould have been within cells, and

not freely floating around in the supernatant.

When all reactor biomasswere harvested via ultrafiltered and

resuspended in fresh basal medium, dechlorination activity

improved markedly in each reactor (Fig. 4A). This could have

been the result of providing each of them the additional B12

associated with the replacement of the medium. However, it is

also possible that along with replacement of beneficial growth

factors, the exchange of medium removed some toxic and/or

inhibiting metabolites accumulated because of the long

hydraulic retention time (i.e., 140 days) adopted in this study.

The beneficial effect of maintaining relatively short hydraulic

retention times (HRT) is supported by the results of our previous

studies (Aulenta et al., 2003) in which full and more stable

dechlorination of PCE (0.5 mmol/L) to ETH with methanol as

electron donor was maintained for over 300 days in a fixed-bed

reactor operated at 10-day HRT.

The greater process stability observed in themethanol- and

butyrate-fed bioreactors could have been due to the esta-

blishment of a more diverse and hence more stable microbial

community in which non-dechlorinating microorganisms

played a role by both providing dechlorinators with growth

factors and being able to cope or partially reduce the toxicity

of accumulatedmetabolites. Froma practical perspective, the

results of this study indicated that the Me- and Bu-reactors

showed a more stable, long-term dechlorinating activity

compared to the H2-reactor, which experienced a progres-

sively diminishing ability to dechlorinate. Nonetheless, the

H2-reactor cumulatively dechlorinated more PCE (in terms

of electrons routed to dechlorination) than other reactors. In

other words, the fraction of reducing equivalents diverted to

methanogenesis in the H2-reactor was less than the fraction

diverted to fermented end-products in the Me- and Bu-

reactors. This was true at least in a cumulative sense over the

500-day operation, inwhich a total PCE supply of 30mmol/L

was administered. In large part, this was probably due to the

fact that 100% of supplied equivalents were in the form of H2

in the H2-fed reactor, whereas a much lesser fraction of

supplied equivalents became H2 in the reactors fed organic

electron donors. Themajority of the equivalents fromorganic

donors became acetate via donor fermentation, and acetate

could not be used as an electron donor by dechlorinators in


these consortia. However, this is not a general conclusion

since the reduction of PCE coupled to acetate oxidation has

been previously described (He et al., 2002; Krumholz et al.,

1996). Therefore, based on the results of this study, direct H2

supply can be considered as a possible alternative when

remedial action can be or has to be performed in a shorter

time frame and/or can be concentrated in an engineered

system, like a biological permeable barrier.

This study has also highlighted the negative effect of a long

hydraulic retention time on the performance of dechlorinat-

ing cultures. In flow-through systems (e.g., biological

barriers) this effect should be probably minimized by the

constant supply of groundwater, nonetheless, it could still

have some relevance in a field system in which contaminated

groundwater is continuously recirculated between extraction

and injection zones.

We are thankful to Giancarlo Minervini and Alessandro Cannone for

maintenance of the bioreactors.

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